Botulinum toxin is a neurotoxic protein produced by the bacterium Clostridium botulinum, as well as other Clostridial species, such as Clostridium butyricum, and Clostridium baraffi. The toxin blocks neuromuscular transmission and causes a neuro-paralytic illness in humans and animals, known as botulism. C. botulinum and its spores commonly occur in soil and putrefying animal carcasses, and can grow in improperly sterilized or improperly sealed food containers, which are the cause of many botulism cases. Botulism symptoms can include difficulty walking, swallowing, and speaking, and can progress to paralysis of the respiratory muscles and finally death.
Botulinum toxin type A is the most lethal natural substance known to man. In addition to serotype A, six other generally immunologically distinct botulinum toxins have been characterized, namely botulinum toxin serotypes B, C1, D, E, F, and G. The different serotypes can be distinguished by neutralization with type-specific antibodies and vary in severity of paralysis they evoke and the animal species they mostly affect. The molecular weight of the botulinum toxin protein molecule, for each of the known botulinum toxin serotypes, is about 150 kD, composed of an about 100 kD heavy chain joined to an about 50 kD light chain. Nonetheless, the botulinum toxins are released by Clostridial bacteria as complexes of the 150 kD toxin with one or more non-toxin proteins. For example, botulinum toxin type A exists as 900 kD, 500 kD and 300 kD complexes (approximate molecular weights).
Despite the known toxic effects, Botulinum toxin type A is clinically used to treat a variety of indications, including, e.g., neuromuscular disorders characterized by skeletal muscle hyperactivity. For example, BOTOX® is the trademark of a botulinum toxin type A complex available commercially from Allergan, Inc., of Irvine, Calif. Botulinum toxin type A finds use, for example, in the treatment of essential blepharospasm, strabismus, cervical dystonia, and glabellar line (facial) wrinkles Other serotypes also have been used clinically. A botulinum toxin type B, for example, has been indicated for use in treating cervical dystonia. The botulinum toxins are believed to bind with high affinity to the presynaptic membrane of motor neurons, translocate into the neuron, and thereafter block the presynaptic release of acetylcholine.
The botulinum toxin for clinical use is typically isolated from cell culture and various purification approaches have been used. Historically, the toxin is purified in complexed form by a series of precipitation and tangential flow filtration steps. See, e.g., Schantz E. J., et al., Properties and use of botulinum toxin and other microbial neurotoxins in medicine, Microbiol Rev 1992 March 56(1):80-99. Such approaches have provided relatively low yields, however, typically less than about 10%. Other approaches have used size exclusion, ion exchange, and/or affinity chromatography. See, e.g., Schmidt J. J., et al., Purification of type E botulinum neurotoxin by high-performance ion exchange chromatography, Anal. Biochem. 1986 July; 156(1):213-219; Simpson L. L., et al., Isolation and characterization of the botulinum neurotoxins, Harsman S, ed. Methods in Enzymology. Vol. 165, Microbial Toxins: Tools in Enzymology San Diego, Calif.: Academic Press; Vol. 165: pages 76-85 (1988); Kannan K., et al., Methods development for the biochemical assessment of Neurobloc (botulinum toxin type B), Mov Disord 2000; 15(Suppl 2):20 (2000); Wang Y. C., The preparation and quality of botulinum toxin type A for injection (BTXA) and its clinical use, Dermatol Las Faci Cosm Surg 2002; 58 (2002); and U.S. Pat. Appl. Publ. No. 2003/0008367.
Still other approaches have focused on just one of the toxin's heavy or light chains, rather than a complete and biologically active botulinum toxin protein. For example, one of the chains is individually synthesized by recombinant means. See, e.g., Zhou L., et al., Expression and purification of the light chain of botulinum neurotoxin A: A single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain, Biochemistry 1995; 34(46):15175-81 (1995); and Johnson S. K., et al., Scale-up of the fermentation and purification of the recombination heavy chain fragment C of botulinum neurotoxin serotype F, expressed in Pichia pastoris, Protein Expr and Purif 2003; 32:1-9 (2003). These approaches, however, require extra steps to reform a complete and biologically active botulinum toxin protein.
A more recent approach involves the use of hydrophobic interaction chromatography, mixed mode, and/or ion exchange chromatography to purify a botulinum toxin as a complex. See, e.g., U.S. Pat. Nos. 7,452,697 and 7,354,740, which are hereby incorporated by reference.
Accordingly, there is a need in the art for improved purification methods for isolating complete botulinum toxin proteins in stable, biologically active, but non-complexed forms. It is therefore an object of the invention to provide compositions and methods addressing these and other needs.
The foregoing discussion is presented solely to provide a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application.